Low molecular weight human, mouse and rat transforming growth factors (TGFs) were isolated from tissue culture media conditioned by: (1) two human metastatic Ormelanoma tumor lines, (2) a Moloney murine sarcoma virus-transformed mouse 3T3 cell line; and (3) two Fischer rat embryo fibroblast cell lines, nonproductively transformed, one by Snyder-Theilen feline sacoma virus and the other by Abelson murine leukemia virus. These TGFs compete for binding to the cellular epidermal growth factor (DGF) receptor. The purification of each TGF was achieved by gel permeation chromatography, followed by reverse phase high pressure liquid chromatography using sequentially acetonitrile and 1-propanol in the presence of aqueous trifluoracetic acid. The amino-terminal sequences of rat, mouse and human TGFs showed extensive sequence homology among polypeptides from different species and cell types. The complete primary structure of rat TGF was determined. Rat TGF is a single chain polypeptide, with a calculated molecular weight of 5,600, which displays 33% sequence homology with murine EGF and 44% sequence homology with human urogastrone. These results emphasize that TGFs which are associated with the control of cell growth and differentiation are highly conserved through vertebrate evolution and are chemically distinct, but are evolutionarily related to EGFs found in normal cells and tissue fluids.